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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
,2,3,5
* Institut National de la Recherche Agronomique, Unité de Pharmacologie-Toxicologie, UR66, 180 chemin de Tournefeuille, 31931 Toulouse Cedex 9, France;
and
Animal Nutrition Group, Wageningen University and Research Centre, PO Box 338, 6700 AH, Wageningen, the Netherlands
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were analyzed using a semiquantitative reverse-transcription PCR technique on d 1, 4, and 10 in the ileum and colon of piglets fed either a test diet (CHO) or a control diet. In addition to the diet, the effect of enforced fasting on cytokine mRNA content was also evaluated. No effect of fasting was observed on the pro-inflammatory cytokine mRNA content. Our results showed that the CHO diet induced an up-regulation of IL-6 mRNA content in the colon of piglets 4 d postweaning. This up-regulation was specific for the animals fed the CHO diet and was not observed in animals fed the control diet. An increase in IL-1ß mRNA content was also observed on d 4 postweaning in all of the piglets. Correlations between proinflammatory cytokines and the end-products of fermentation indicated that the regulation of cytokines may be linked with some of the fermentation end-products such as branched-chain fatty acids, which are in turn end-products of protein fermentation.
Key Words: pig • prebiotic • proinflammatory cytokine • weaning
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Awati and Moughan, 2006). Prebiotics include short-chain carbohydrates, such as inulin and fructo-oligosaccharides, that are nondigestible by human digestive enzymes but that can be metabolized by colonic bacteria to produce short-chain fatty acids (SCFA), such as butyrate.
In the pig, several investigators have shown that the inclusion of dietary fermentable carbohydrates can modulate the gastrointestinal microbiota by increasing the growth of lactic acid bacteria (Houdijk et al., 2002; Smiricky-Tjardes et al., 2003; Konstantinov et al., 2004). Proliferation of endogenous bacteria such as bifidobacteria may inhibit colonization of the intestine by pathogens, and a diet supplemented with prebiotics has been shown to prevent infections in pigs (Leser et al., 2000; Letellier et al., 2000).
Apart from their effect on the intestinal microbiota, little is known concerning the possible implications of prebiotics on the host response, especially in the pig. It has been demonstrated recently that weaning is associated with increased proinflammatory cytokine mRNA content in the intestine of piglets (Pié et al., 2004).
The current study was designed to examine the effects of inclusion of fermentable carbohydrates in the diet of weaning piglets on the fermentation end-product profile and on mRNA contents of 6 proinflammatory cytokines, which are indicators of inflammatory processes in the gut, to determine whether there is any correlation among these variables. This was an attempt to elucidate how microbial activity in terms of end-products can influence the inflammatory process in the gut to design diets that could affect the gut inflammatory status by controlled stimulation of the fermentation process.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental Design
The experiment was designed as 2 x 2 factorial arrangement of treatments, with 2 diets and 2 feeding treatments. The experiment was conducted in 2 identical replicates. For each replicate, 9 piglets were chosen randomly from each of the 4 litters (36 piglets per replicate, and 72 piglets in total for the experiment).
On d 1 of each period, 1 piglet from each litter was killed (4 piglets), and tissue and digesta samples were collected, before the other piglets were subjected to any of the treatment combinations. On d 4 and d 10, from each litter, 1 piglet from each of the 4 treatments was killed for collection of tissue and digesta samples from the terminal ileum and the colon (Figure 1). These time points were chosen because previous studies had demonstrated that weaning is associated with a transient up-regulation of inflammatory cytokine mRNA content on d 3 to 4 after weaning and that the levels of most cytokines rapidly return to preweaning values at d 9 after weaning (Pié et al. 2004).
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Dietary Treatments
The composition of the diets is shown in Table 1. The control diet was designed to have very low levels of fermentable carbohydrates. The test diet with added fermentable carbohydrates (CHO) was based on this same diet, but had added carbohydrates in the form of unmolassed sugarbeet pulp (pulp with most of the molasses removed), native wheat starch, lactulose, and inulin. The diets were composed in such a way that GE and CP contents were comparable. Neither diet contained antibiotics or copper beyond that of the trace mineral premix.
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Slaughtering and Sampling
The piglets were slaughtered on d 1, 4, and 10. First, ketamin (Sanaket 10%, Anisane B.V., Raamsdonksveer, the Netherlands) was used as a preanesthetic (15 mg/kg of BW), and 30 min later, the piglet was euthanized by intracardiac injection of T61 (combination of embutramide, mebenzoniumiodide, and tetracain hydrochloride; Hoechst Roussel Vet, Frankfurt, Germany). After abdominal dissection, plastic strips were used to seal off random areas of the intestine, to reduce mixing of digesta before the entire GIT was separated from the abdominal cavity.
At necropsy, tissue samples were collected from the terminal ileum and the colon. Digesta particles were removed from the terminal ileum and colon segments by flushing with a cold saline solution. Samples were stored at –20°C pending analysis. The tissue segments were placed in 1 mL of Extract-all (Eurobio, Les Ulis, France), frozen in liquid nitrogen, and stored at –80°C before mRNA analysis. Care was taken to keep the time between death of the piglet and tissue collection at <10 min.
Tissue Sample Analyses, RNA Extraction
Intestinal samples were homogenized using a Cat homogenizer (Labomoderne, Paris, France). Total RNA was extracted with a monophase solution of phenol and guanidine isothiocyanate, following the manufacturer’s recommendations. Briefly, after the homogenization step the samples were mixed with chloroform (20%, vol/vol) and shaken vigorously. The suspension was centrifuged (12,000 x g for 15 min). The aqueous phase was mixed with an equal volume of isopropanol, and the RNA was then pelleted by centrifugation and washed with 75% ethanol. The RNA was resuspended in 50 µL of ultrapure water containing 0.02% (wt/vol) diethyl pyrocarbonate (Sigma, St. Quentin Favallier, France). Total RNA was quantified using a spectrophotometer at a wave length of 260 nm, and the purity was assessed by determining the ratio of absorbance at 260 and 280 (A260/A280). All samples had a ratio ranging between 1.7 and 1.9. The quality of the RNA was verified by electrophoresis of the samples on agarose gels containing ethidium bromide and examination of the presence of the 18S and 28S ribosomal bands.
Reverse-Transcription PCR Detection of Cytokine mRNA and Quantification of PCR Products
Semiquantitative determination of tumor necrosis factor- (TNF-), IL-1ß, IL-6, and cyclophilin was carried out using reverse-transcription PCR, as previously described (Dozois et al., 1997; Fournout et al., 2000; Marin et al., 2002), with minor modifications. As a first step, 1.5 µg of total RNA was reverse transcribed using random hexamers (Boehringer Mannheim, Meylan, France) and murine Moloney leukemia virus reverse transcription (Point Mutant, Promega, Charbonnières, France).
After reverse transcription, the resulting cDNA were amplified by PCR using deoxynucleoside triphosphates (2 mM each; Eurobio), 0.2 pM 5'- and 3'-primers, 2 mM MgCl2, and 2.5 U of DNA Taq polymerase (Invitrogen, Cergy Pontoise, France) in a final volume of 50 µL. The PCR primers and the annealing temperatures used for the amplification were the same as previously described (Dozois et al., 1997). The number of PCR cycles for each transcript was as follows: 30 cycles for TNF-, IL-1ß, and IL-8; 35 cycles for IL-6, IL-12p40, and IL-18; and 24 cycles for cyclophilin. Twofold dilutions of representative samples were used to verify that the amplification conditions were nonsaturated and suitable for semiquantitative analysis.
In a second step, semiquantitative analysis of all PCR products was done by hybridization of 33P-labeled, specific oligonucleotide probes to the PCR products immobilized on nitrocellulose membranes by dot blotting, as previously described (Darwich et al., 2003; Techau et al., 2007). The DNA probes used for hybridization of the different cytokines were already described (Pié et al., 2004). The relative amounts of each product were determined by measuring radioactivity with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For each cytokine, the relative amount of cytokine mRNA was normalized to cyclophilin mRNA. The data were further normalized against the control animals (considered as 1 AU) by the following formula:
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Digesta Sample Analyses
Digesta were analyzed for DM, pH, and SCFA (including lactic and formic acids in the small intestine) as previously described. Dry matter was determined by drying to a constant weight at 103°C (ISO 6496: ISO, 1999), and ash was determined by combustion at 550°C (ISO 5984: ISO, 1978). The VFA concentrations were analyzed by gas chromatography (Fisons HRGC Mega 2, CE Instruments, Milan, Italy), using a glass column fitted with Chromosorb 101 (Supelco, Zwijndrecht, the Netherlands), N2 saturated with methanoic acid as the carrier gas at 190°C, and iso-caproic acid as an internal standard (Awati et al., 2005). Ammonia and lactic acid concentrations were determined according to the method described by Houdijk et al. (2002) and Voragen et al. (1986), respectively. Previous studies had shown that the lactic acid concentrations were low in the colonic contents (unpublished data), so this variable was measured only in the ileal contents.
Statistical Analysis
The results were expressed as mean ± SD. Statistical analyses were performed using ANOVA, as already described (Awati et al., 2006). The following statistical model was used:
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where Y is the dependent variable to be tested; µ is the overall mean; Di represents the effect of the diet i; Fj is the effect of the fasting treatment j; (D x F)ij is the interaction; 
1ijk is error term 1, which represents the random effect of animal within diet i and fasting treatment j; Gl represents the effect of site of GIT l; (D x G)il, (F x G)jl, and (D x F x G)ijl denote the respective interactions; and 2ijklm is error term 2, which represents the overall error. Fisher’s test was used to examine significant differences between treatment groups. Differences were considered significant when P < 0.05. To examine correlations between each cytokine in relation to digesta measurements, a stepwise multiple regression analysis was done using PROC REG (SAS Inst. Inc., Cary, NC).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and 3). A significant increase in IL-1ß mRNA was observed in the colon at d 4 post-weaning in animals fed the control diet (P = 0.04) and the CHO diet (P = 0.0002), compared with the d-1 values (Figure 2). Then by d 10, for both groups of piglets, the mRNA expression levels of IL-1ß returned to levels similar to those obtained on d 1. A kinetic profile similar to those observed for IL-1ß mRNA was also obtained for the IL-6 cytokine in animals fed the CHO diet. Indeed, a transient up-regulation of IL-6 mRNA (P = 0.004) was observed on d 4 postweaning in the colon of piglets fed the CHO diet (Figure 2). By contrast, no increase of IL-6 mRNA between d 1 and 4 was observed for the group of piglets fed the control diet. For TNF- mRNA, a decrease (P < 0.05) was observed on d 10 postweaning compared with d 1 and 4 postweaning for both groups of animals. A decrease (P < 0.05) of IL-12p40 and IL-18 mRNA content was found on d 10 postweaning compared with d 1. Although this decrease was significant only in replicate A, the same trend was observed in replicate B (data not shown). No changes in the amounts of IL-8 mRNA were found between piglets fed the control and CHO diets (2.24 and 2.51 at d 4; 1.55 and 1.22 at d 10 for piglets fed the control and CHO diets, respectively).
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were also measured in the ileum (Figure 3
). An increase in IL-1ß was observed on d 4 postweaning in this part of the gut in animals fed the control diet (P = 0.04) and the CHO diet (P = 0.04). No change in IL-6 mRNA level was observed for either diet treatment. No change was observed for the level of TNF- mRNA (Figure 2). An analysis of IL-8, IL-12p40, and IL-18 mRNA content were also performed because we had already demonstrated that weaning could modulate the level of these cytokines (Pié et al., 2004). No significant alteration of the level of these cytokines in piglets fed the control diet and the CHO diet were observed (data not shown).
Influence of Fasting on the mRNA Content of Proinflammatory Cytokines at Weaning
Low voluntary feed intake, a characteristic often occurring in piglets just after weaning, can also modulate gut inflammation. For this reason, a comparison was also made of the profiles for proinflammatory cytokine mRNA expression on d 4 postweaning in 2 groups of piglets: 1 group fasted for 2 d postweaning and the other group given free access to feed. Not surprisingly, total feed intake during the first 4 d post-weaning was lower (P < 0.05) in the group of fasted piglets than in the group of nonfasted piglets (Table 2). However, a comparison of cytokine levels on d 4 postweaning did not show any differences between the fasted and nonfasted piglets fed the control diet or the CHO diet (Table 2). Similar results were obtained for the ileum (data not shown). Fasting also had no influence on the cytokine mRNA content measured at d 10 after weaning in the ileum and the colon of piglets fed the control or the CHO diet (data not shown).
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). Total VFA; acetic, propionic and butyric acids; and ammonia concentrations all increased with time, whereas DM, lactic acid, and formic acid concentrations decreased. Differences between the GIT sites were highly significant (P < 0.001) for all variables except lactic acid and formic acid. Volatile fatty acid concentrations, ammonia concentrations, DM, and BCP were greater in the colon. However, surprisingly, pH was lower in the ileum compared with the colon (Table 3). Diet also influenced these variables; VFA (P < 0.01) (except butyric acid) and lactic acid concentrations (P < 0.1) were greater in the CHO diet, whereas the BCP and ammonia concentration were less (P < 0.001) in this diet compared with the control diet. Fasting had an effect on ammonia and the propionic, acetic, and lactic acid concentrations (P < 0.05). There were lower ammonia and lactic acid concentrations, coupled with greater propionic and acetic acid concentrations for the nonfasting compared with fasting animals.
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). There was also an interaction (P < 0.01) between diet and GIT site for propionic acid, DM, pH, and ammonia concentrations.
Correlation Between Proinflammatory Cytokine mRNA Content and Digesta Measurements
Tables 4 and 5 show the correlations obtained using step-wise regression for the 6 cytokines correlated with DM, pH, and fermentation end-products in the piglet digesta. Table 4 shows the correlations obtained from the colon samples, and Table 5, those for the terminal ileum.
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. In addition, IL-8 correlated negatively (P = 0.043) with butyrate, whereas IL-12p40 (P = 0.004) and IL-18 (P = 0.026) had a negative correlation with BCP and acetic acid, respectively. No cytokines in colonic tissue were correlated with ammonia or propionic acid. For the terminal ileum, the number of digesta measurements that were correlated with cytokines was greater than for the colon. In this case, ammonia had a negative correlation with IL-1ß (P < 0.1), IL-6 (P < 0.05), and also with IL-8, although not a significant one. Dry matter, butyrate, BCP, and lactic acid, when correlated with most cytokines, had a positive influence. The pH and acetic acid were not correlated with any of the cytokine values.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In a recent study, Pié et al. (2004) demonstrated that weaning is associated with an early inflammatory cytokine response in the intestine of newly weaned piglets. Because diet can modulate the inflammatory response in the gut (Kudsk, 2003), the current study investigated the effect of a diet supplemented with indigestible but fermentable carbohydrates on the intestinal level of a set of proinflammatory cytokines, and then correlated these values with variables obtained for digesta chemistry. These results showed that the diet enriched with CHO induced an up-regulation of IL-6 mRNA level in the colon of piglets at 4 d postweaning. An increase in IL-1ß mRNA was similarly observed at d 4 postweaning in piglets fed the CHO diet, but also in pigs fed the control diet.
Many studies have shown that diet can influence the immune response (Cunningham-Rundles, 2001), so this factor may represent one important influence on the regulation of GIT inflammation in newly weaned animals. In the current study, a significant up-regulation of IL-6 mRNA was observed in the colon of weaned piglets fed the CHO diet, but not in piglets fed the control diet, indicating that the regulation of IL-6 mRNA in the colon depends on dietary factors, or at least on microbiological factors that have been influenced by diet. Short-chain fatty acids, which are predominant end-products of dietary carbohydrate fermentation in the colon, and which can increase IL-6 abundance in the intestine of piglets (Milo et al., 2002), represent a good candidate that could modulate IL-6 mRNA content in the colon of piglets. However, comparisons of IL-6 mRNA contents with SCFA (acetic, propionic, and butyric acids) concentrations in the ileum and colon of weaned piglets showed no correlation between the levels of IL-6 mRNA and SCFA, indicating that the increased level of this cytokine was not directly dependent on the presence of SCFA. On the other hand, these results also demonstrated that lactic acid, in the ileum, was positively correlated with IL-6 mRNA. This may indicate that the IL-6 mRNA content is linked in some way to bacterial fermentation, most probably by lactobacilli or other lactic acid-producing species, at least in the ileum. In agreement with this finding, other studies have shown that non-pathogenic bacteria such as lactic acid bacteria can induce the production of cytokines such as IL-6 (Miettinen et al., 1996; Morita et al., 2002a,b). Because carbohydrates favor the growth of these bacteria in the colon, it is possible that the increase of IL-6 mRNA observed in piglets fed the CHO diet resulted from stimulation of specific bacterial species. Further studies are needed to elucidate the pathway by which carbohydrates could modulate IL-6 content.
The increase in IL-6 mRNA level in animals fed the CHO diet was surprising. Indeed, several reports have indicated that prebiotics tend to suppress inflammatory responses rather than the reverse (Kanauchi et al., 1998; Videla et al., 2001; Cherbut et al., 2003). However, in the case of IL-6, it is noteworthy that this cytokine, first regarded as proinflammatory, also has other properties. In particular, this cytokine plays an important role in the terminal differentiation of B cells to antibody-producing plasma cells, and in the driving of naïve CD4+ T cells into effector Th2 cells (Van Snick, 1990). Furthermore, IL-6 is known to have antiinflammatory activities, such as inhibition of macrophage TNF and IL-1 release (Akira et al., 1990; Van Snick, 1990).
In addition to IL-6, analysis of IL-1ß mRNA content in weaned piglets also demonstrated that this cytokine was up-regulated by 3 d postweaning. However, this up-regulation was similar in piglets fed the CHO diet and the control diet, suggesting that the level of this cytokine was not influenced by the diets used. In support of this hypothesis, analysis of IL-1ß mRNA in relation to dietary factors did not show that the level of this cytokine, both in the colon and the ileum, was correlated with any of the end-products of fermentation (except for ammonia in the ileum, but the probability of correlation was low). In agreement with these results, an increase in IL-1ß concentration was also observed in the blood of weaned piglets independent of the diet (McCracken et al., 1999). It is noteworthy that in a previous study (Pié et al., 2004), in which different experimental conditions were used, there was also a rapid mRNA increase in IL-1ß after weaning. This may indicate that an increase in IL-1ß mRNA is a common gut phenomenon at the time of weaning, and may be considered to be a general and early immunological change associated with weaning (or other stress) in piglets.
In the ileum, a positive correlation was observed between BCP and IL-8, IL-12p40, and IL-18 mRNA, indicating that the products of protein fermentation may favor inflammatory responses at weaning. In contrast, no correlation between these inflammatory cytokines and BCP was found in the colon. The lymphoid structures, Peyer’s patches, present in the ileum but not in the colon (Pabst and Rothkotter, 1999), may explain the variation in the responses observed between these 2 areas of the GIT.
Despite a strong correlation between IL-8, IL-12p40, and IL-18 mRNA and BCP in the ileum, no postweaning changes in the mRNA content of these cytokines were observed in piglets fed the CHO or the control diet. This may indicate that the proportion of branched-chain fatty acids, increased as a result of protein fermentation, were not sufficient to induce significant changes of the cytokine level or that other factors may be implicated in the regulation of these cytokines. In the colon, a positive correlation between DM and the cytokines IL-12p40, IL-18, and TNF- was also obtained. Dry matter, which reflects movement of the colonic water, is affected by dietary factors such as fiber (Low et al., 1978) and SCFA (Ruppin et al., 1980) and by microbial factors including enteric bacterial toxins (Sears and Kaper, 1996). Because these factors are also potential inducers of proinflammatory cytokines, the DM content may represent a coincidental indicator of some inflammatory responses in the colon of weaned piglets.
In addition to the diet, the low feed intake commonly observed immediately after weaning in piglets may represent an important factor that can influence the cytokine response. Indeed, recently it has been reported that lack of intestinal nutrient provision (i.e., parenteral feeding) favors the development of inflammation in the intestine of piglets (Ganessunker et al., 1999; Zijlstra et al., 1999). McCracken et al. (1999), who examined numbers of inflammatory T cells during the weaning period, suggested that impaired feed intake immediately after weaning may contribute to inflammation in the intestine of piglets. In the current study, however, there was no correlation between the quantity of feed intake and the mRNA content of the proinflammatory cytokines IL-1ß, TNF-, and IL-6 by d 4 postweaning. Indeed, up-regulation of IL-1ß and IL-6 mRNA was observed on d 4 postweaning at the same levels in animals fasted for 2 d after weaning as for the control, nonfasted animals. For IL-6, this is in agreement with other results, which may indicate that this cytokine is regulated by dietary factors. For IL-1ß, for which the level seems to be independent of the diet and feed intake, it was demonstrated in a previous study that IL-1ß mRNA was increased in nearly all parts of the intestine (Pié et al., 2004). Together, these results strongly support our previous hypothesis that the up-regulation of IL-1ß mRNA is probably related to the general stress associated with weaning. In support of this hypothesis, Kanitz et al. (2004) recently demonstrated that social stress (i.e., maternal deprivation and social isolation) during the neonatal period resulted in an increase in IL-1ß in the brain of piglets. Alternatively, the up-regulation of IL-1ß or TNF- mRNA or both could be the result of an increased resorption of lipopolysaccharide from the intestine, because of an alteration of the intestinal barrier at weaning (Boudry et al. 2004). Indeed, lipopolysaccharide is a well-known inducer of inflammatory cytokines in all animal species, including pigs (Touchette et al., 2002). Local inflammation caused by weaning anorexia (McCracken et al., 1999) may also contribute to the observed changes.
In conclusion, our results demonstrate that a diet enriched in fermentable carbohydrates can modulate the IL-6 mRNA content during the weaning period in piglets. Correlations between several proinflammatory cytokines and the end-products of fermentation demonstrated that the regulation of cytokines is strongly linked to fermentation end-product measurements such as BCP and ammonia, particularly in the ileum. Cytokines play a major role in the maintenance of gastrointestinal homeostasis, and their regulation by nutritional factors may be an important consideration in the future for the development of new diet formulas for newly weaned piglets.
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Footnotes |
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2 These authors contributed equally to this work.
3 Present address: Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
4 Present address: Centre for Nutrition and Food Sciences, University of Queensland, St. Lucia, Qld. 4072, Australia.
5 Corresponding author: ioswald{at}toulouse.inra.fr
Received for publication August 4, 2006. Accepted for publication November 6, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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